This is an R01 renewal proposal to study immune tolerance using custom designed super-antigens (Ags) reactive to immunoglobulin (Ig) constant regions. Immunological tolerance regulates unwanted responses of B and T cells to self- tissue, reducing the chances of autoimmunity and focusing the immune response on foreign antigens. SuperAgs facilitate the analysis of tolerance among normal, polyclonal populations of cells. In the present proposal, we make use of two transgenic models, one ubiquitously expressing superAg to IgM (IgM-macroself) and a second expressing in the liver a superAg to immunoglobulin ? light-chain (pAlb mice). IgM-macroself mice are a model of central tolerance in which B cell development is blocked at the immature B cell stage in the bone marrow and peripheral B cells are absent. In the pAlb model, B cells carrying ? L-chain are deleted in the periphery at a later, still semi- mature CD93+ transitional stage and fail to populate lymph nodes. Previous work has suggested that tolerance mechanisms at these checkpoints are distinct. We established a transposon insertional mutagenesis system to generate and identify mutant B cells in vivo that have escaped superAg-mediated tolerance. We identified a number of potential candidate genes and verified one candidate whose mutation allowed B cell escape. We propose to validate candidate genes regulating these two tolerance checkpoints, find additional candidates, evaluate in depth the role of Caspase 2, and investigate the use of genetic reprogramming to genetically capture escaped B cells and their mutations. The long-term goal of these studies is to develop a comprehensive understanding of all of the genetic components maintaining immune tolerance at these stages. This knowledge should facilitate treatment of autoimmune disorders and impact vaccine development.